Although the Nef protein of HIV-1 has been the most enigmatic and controversial of the viral auxiliary proteins, work in the SIV system strongly suggests that it is likely to be critically important for both viral replication and pathogenesis in vivo. Recently, it has become evident that both HIV-1 and SIV Nef can induce an efficient and specific post-translational down-regulation of the cell-surface expression of the CD4 glycoprotein receptor for these primate lentiviruses. In this grant application, we are proposing a series of experiments that are designed to clarify the mechanism by which this specific down-regulation is accomplished and to reveal whether this effect can explain the critical importance of Nef for viral replication in vivo when compared to its dispensability for replication in culture. We will, in particular, identify the sequences in CD4 that are necessary and sufficient to mediate down-regulation by Nef and attempt to define functional domains within Nef itself by targeted mutagenesis. These reagents will then be used in experiments that are designed to address the possibility of a direct interaction between CD4 and Nef or a Nef-induced cellular co-factor. We will also address the consequences of HIV-1 and SIV Nef expression for T- cell receptor mediated signal transduction in CD4+ T-cells and attempt to develop culture conditions that recreate the marked positive effect of Nef on viral replication in vivo. These latter experiments will, in particular, be designed to test the hypothesis that the primary role of Nef is to enhance viral spread by inhibiting the reinfection of infected CD4+ T-cells by released progeny virions. Overall, we believe that the research outlined in this proposal has the potential to significantly increase our understanding of not only the mechanism of action but also the biological role of the Nef proteins of HIV-1 and SIV.